The use of lanthanide chelates in nonisotopic labelling of biological macromolecules has attracted a great deal of interest in the field of diagnostics This technique takes advantage of the long lived fluorescence of lanthanide elements, compared to ordinary fluorescent backgrounds which otherwise tend to overwhelm genuine signal. The trivalent lanthanide ions Eu.sup.+++, Tb.sup.+++, and Sm.sup.+++ all have fluorescent decay times on the order of milliseconds compared to nanosecond decay times for background fluorescence. By irradiating a sample at the appropriate wave-length and energy level, the fluorescence may be measured at a delayed point in time, after background fluorescence has already decayed, but while the lanthanide specimen is still emitting This technique is known as time-resolved, or time-gated fluorescence spectroscopy. For a general review of the principles of the technique, see U.S. Pat. Nos. 4,150,295 and 4,058,732, and Immunoflurescence and Related Staining Techniques, Knapp, et al. eds. (1978: Elsefier/North Holland Biomedical Press).
The fluorescent properties of lanthanide ions are generally enhanced when they are captured by a chelating agent. This is because hydration of the ion in aqueous solution drastically quenches the emitted energy. Chelation is also necessary to entrain the ions in proximity to the target which they are to detect.
Covalent coupling of chelating agents to proteins, such as antibodies with binding specificity to a targets and to nucleic acids, which will hybridize to specific complementary nucleic acid sequences, is known in the art. For example, WO 89/04375 (Musso) discloses labelling of DNA probes with EDTA, DTPA, and certain analogs thereof such as p-phenyl-EDTA, through a linker moiety having a terminal group with the formulae NH(C.dbd.S)NH, NH(C.dbd.O)NH, S(C.dbd.S)NH, etc. The probes are complexed with lanthanide ions and utilize a beta-diketone in micelles to increase sensitivity. Similarly, WO 88/02784 (Ylikoski) discloses multiple chelate labelled polymeric probes having chelate moities of the modified EDTA and DTPA type. WO 90/00550 (Kankare) discloses novel terpyridine derivatives which act as chelating agents for lanthanide ions and may have utility in labelling nucleic acid probes and proteins.
EP 0 324 323 (Hemmila) discloses chelates having a structure containing a heteroatom having a free electron pair selected from nitrogen, oxygen, phosphorus or sulfur, and being bonded so that the free electron pair is delocalized to a conjugated system of pi-bonds, useful in a homogeneous assay format WO 90/00623 (Kwiatkowski) discloses a multilabel nucleic acid probe system utilizing chelates having a bipyridine structure as a vehicle for multiple dicarboxylic acid groups.
The properties of various chelating agents differ with the type of biological macromolecule to which they are attached Of particular interest are the poly(arylpyridine) chelates which have large capacity to bind ions. The monosubstituted amino triazine ligand molecule having two diacid groups binds rare earth ions efficiently then attached to proteins such as antibodies, but has serious limitations in probe assays. In the context of protein labelling, the ligand combines at a number of sites which confer a conformational configuration conducive to ion binding. Naturally occuring carboxyl groups, such as the free carboxyl group of glutamic acid, also promote chelation. However, no such conformational interactions are possible with nucleic acids, which behave much like linear molecules. Thus, it is not readily apparent, nor can it be predicted, which chelate structures have especial efficacy in nucleic acid probe assays.
SUMMARY OF THE INVENTION
The present invention relates to novel lanthanide chelate-conjugated oligonucleotides utilized in hybridization assays for detection of nucleic acids present in a sample in small amounts, frequently in the presence of large quantities of non-homologous nucleic acids. Et is therefore an object of the present invention to obtain chelates capable of being conjugated covalently to oligonucleotides, which have extremely high level rare earth element capture efficiency, and corresponding high emission levels This is especially important in labelling of nucleic acids since indiscriminate multiple chelate labelling of nucleotide bases interferes with binding specificity.
Another object of the present invention, is to provide chelates which may be universally coupled to any nucleic acid sequence. The present chelates have an oligonucleotide tail of at least 4 nucleotides adapted for hybridization to a separate partially complementary nucleic acid strand. Upon hybridizing, a new strand complementary to that hybridized to the oligonucleotide tail may be synthesized by invitro enzyme-catalyzed polymerization utilizing the tail as a primer. Alternatively, a non-complementary strand can be incorporated into the chelated oligonucleotide by first hybridizing both the strand and the oligonucleotide to a bridging sequence, and then ligating. Alternatively, the oligonucleotide and a probe sequence can be blunt-end ligated with RNA ligase.
In accordance with the present invention, rare earth chelate-conjugated oligonuclectides have a rare earth chelate portion comprising one or a plurality of 2-alkoxy-4,6-di(N,N,N',N'-tetraalkyl)amino triazines in which one triazine carbon is covalently linked to a functionalized arylalkyl group selected from the group consisting of p-aminophenethoxy, p-isothiocyanophenethoxy, and p-thionylchlorophenethoxy, and the second and third triazine carbons are covalently coupled to an (aryl-dicarboxylpyridine)alkyl group; a linking group selected from the group consisting of an amide and a thiourea; and an oligonucleotide containing at least 4 consecutive underivatized deoxy- or ribo- nucleotides joined to the rare earth chelate through the linking group.
In another aspect of the present invention, the present chelate-conjugated oligonucleotides may be utilized in an assay for detecting a complementary nucleic acid in which the oligonucleotides are hybridized to a target nucleic acid sequence, followed by separating the hybridized nucleic acids, and detecting the extent of hybridization by measuring the signal generated by a signal means. The improvement in the assay comprises the oligonucleotide probe having at least 12 consecutive underivatized nucleotides having a nucleotide sequence complementary to a sample target sequence, which is linked covalently to a rare earth chelate comprising a tetra(arylpyridine) ligand having a tetramer of substituted arylpyridine diacid units attached covalently to a 2-alkoxy-4,6-diamino triazine radical.
Chelate-conjugated probes of any desired sequence can readily be constructed by hybridizing an oligonucleotide of at least 4 nucleotides which incorporates at the 3' or 5' terminus a rare earth chelate comprising a tetra(arylpyridine) having a tetramer of substituted arylpyridine diacid units covalently attached to a 2-alkoxy-4,6-diamino triazine radical, to a probe having a terminal sequence of at least 4 nucleotides complementary to the oligonucleotide, priming a polymerase-catalyzed extension reaction with the hybridized oligonucleotide, carrying out the extension reaction, separating the strands of the duplex, and isolating the chelate-tagged nucleic acid probe. Equivocally, the complementary strand may be removed by enzymatic digestion.
Alternatively, a chelate-tagged probe of any desired sequence can be constructed in a method comprising providing a bridging sequence complementary to an oligonucleotide of at least 4 consecutive underivatized nucleotides incorporating at the 3' or 5' terminus thereof, a rare earth chelate portion comprising a tetra(arylpyridine) ligands having a tetramer of substituted arylpyridine diacid units covalently attached to 2-alkoxy-4,6-diamino triazine radicals, which is also complementary to the terminus of a probe sequence of opposite 5'-3' polarity, hybridizing the bridging sequence to the oligonucleotide and the probe sequences and ligating the probe sequence to the oligonucleotide.
In a further embodiment of the present inventions a kit is provided for preparing chelate-tagged nucleic acid probes of any sequence incorporating a rare earth chelate-conjugated oligonucleotide comprising a first vessel containing an oligonucleotide of at least 4 nucleotides incorporating at the 3' or 5' terminus, a rare earth chelate comprising a tetra(arylpyridine) ligand having a tetramer of substituted arylpyridine diacid units covalently attached to a 2-alkoxy-4,6-diamino triazine radical, and a second vessel containing an enzyme selected from the group consisting of a polymerase and a ligase.